Infection-induced extracellular vesicles evoke neuronal transcriptional and epigenetic changes

Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammation, and behavior, yet it remains elusive how these changes come about. In this study we investigated how norepinephrine levels are altered by infection. TINEV (Toxoplasma-induced neuronal extracellular vesicles) isolated from infected noradrenergic cells down-regulated dopamine ß-hydroxylase (DBH) gene expression in human and rodent cells. Here we report that intracerebral injection of TINEVs into the brain is sufficient to induce DBH down-regulation and distrupt catecholaminergic signalling. Further, TINEV treatment induced hypermethylation upstream of the DBH gene. An antisense lncRNA to DBH was found in purified TINEV preparations. Paracrine signalling to induce transcriptional gene silencing and DNA methylation may be a common mode to regulate neurologic function.

Strategy for identifying paracrine signalling in DBH down-regulation. Initially, it was considered that several different factors could explain the disproportionate decrease in DBH expression (and hence NE) relative to the small percent of parasitised cells during chronic infection (e.g. neuroimmune responses). As decreased NE and DBH down-regulation (relative to other genes) has been observed in vitro with noradrenergic cell lines, mechanisms other than the host immune system are involved although host cell immunity remained a possibility. A nuclear run-on assay was performed to assess whether the DBH down-regulation was at the transcriptional or post-transcriptional level. De novo transcription in nuclei isolated from infected cell cultures was measured by immunocapture of incorporated biotin-UTP. Lower amounts of nascent DBH mRNA (relative to standards) were found in host cell nuclei from infected than uninfected noradrenergic cell cultures (21 ± 1.6fold, p = 0.00062) ( Fig. 2A). Similarly, de novo transcription of DBH was downregulated in infected human noradrenergic cells (21 ± 1.7-fold, p = 0.032) (Fig. 2B). Hence, infection induced transcriptional gene silencing (TGS) of DBH in human and rat noradrenergic cells.
We then investigated the spreading of DBH down-regulation by examining uninfected cells in cultures to determine whether cells exposed to infected cells were also suppressed in NE as this could help explain the large change in expression observed. A transwell system was used that permits uninfected cells to be exposed to T. gondii-infected cell products. This method was chosen because it differentiates between diffusible signals and parasites injecting components into cells without invasion, as was observed with a Cre/loxP assay in infected mouse brains 36 . DBH expression was measured in uninfected rat noradrenergic cells in the bottom reservoir of the transwell system with the top reservoir containing an infected culture (Fig. 2C). DBH expression in the cells exposed to infected cultures was found to be down-regulated (7.9 ± 2.8-fold, p = 0.02) suggesting that a transmissible factor was released from infected cells. This was subsequently identified as EVs. In contrast, noradrenergic cells that were exposed to cultures containing heat-killed T. gondii ('mock-infected') were unchanged in DBH expression (Fig. 2D). Exposure of a human neuronal cell line to infected cells in the transwell system induced a larger decrease in DBH expression (37± 16-fold, p = 0.0038) than the rat cell line (Fig. 2E). The observed DBH down-regulation is likely to be a minimal baseline as it remains possible that vesicles may stick to the transwell membrane and that EV passage is restricted. Parasite restriction to the upper reservoir of the 0. 4  www.nature.com/scientificreports/  www.nature.com/scientificreports/ reservoirs and monitoring propagation (data not shown). Transwells were set up with infected fibroblasts in the top reservoir and uninfected noradrenergic cells in the bottom reservoir to assess whether the down-regulation was cell-type specific (Fig. 2F). The noradrenergic cells exposed to T. gondii-infected fibroblast cultures were unchanged in DBH gene expression. A further indication that EVs were the permeable effector responsible for the DBH down-regulation was finding that the insoluble components, separated from soluble factors by ultracentrifugation, contained the DBH down-regulating activity in preliminary tests (data not shown). This provided the rationale for EV isolation and testing.
Epigenetic changes associated with DBH down-regulation during infection. As our findings indicated that TGS was responsible for DBH expression changes, the epigenetic state of the DBH gene was investigated 37 . Methylation Sensitive Restriction Enzyme qPCR (MSRE-qPCR) was used to monitor DNA methylation levels in the DBH gene's upstream region where the majority of CpGs are clustered (Fig. 3A). Methylation in the DBH upstream region rose from 16 ± 4.6% to 66 ± 3.8% in infected cultures of noradrenergic cells during the course of the infection ( Fig. 3B; p = 0.00072). As the noradrenergic cells are sensitive to pH changes (ie. neurotransmitter synthesis and synaptic transmission affected) 38,39 , alkaline-shocked tachyzoites were used Cells are orange, parasites red, signalling factors in vesicles (green) and soluble (yellow). (D) DBH expression measured using RT-qPCR (relative to GAPDH) in PC12 cells from the bottom layer with either uninfected control, T. gondii infected, or mock-infected cells in the upper transwell layer. Mock-infected cells were incubated with heat-killed T. gondii tachyzoites n = 3, *p < 0.05. One-way ANOVA p = 0.017, Tukey's post hoc test uninfected and mock-infected vs infected p = 0.020 and p = 0.033, respectively. (E) Noradrenergic BE(2)-M17 cell expression of DBH mRNA cultured in transwells and treated as in (C); n = 3, p = 0.0038. (F) Infected fibroblasts in the upper layer of the transwell system. DBH expression levels were measured in exposed noradrenergic cells. DBH gene expression was not significantly altered in cells exposed to infected fibroblasts (p = 0.84); ± SEM shown, n = 3, Student's t test.  40 . This procedure elevated expression of bradyzoite markers BAG1 and SAG4 (Fig. S5). DBH methylation was also increased in infected human noradrenergic cultures (2.8-fold; range 2.3-4.8-fold; p = 0.0011) with a time-dependent increase in methylation in the region profiled (Fig. 3C).
As an indicator of EV involvement in the epigenetic changes, cells were treated with an inhibitor of EV biogenesis in the transwell system to restrict parasites and parasite-infected cells from contact with the uninfected cell culture layer. The cells exposed to infected cultures had > 50% higher methylation of the DBH promoter (Fig. 3D). These cultures were treated with GW4869, a sphingomyelinase inhibitor that disrupts vesicle budding required for endosomal formation. Addition on GW4869 to the transwell system abrogated the DBH hypermethylation observed in uninfected noradrenergic cells exposed to infected cultures. Treatment with GW4869, a neutral sphingomyelinase (N-SMase) inhibitor, disrupted the DBH promoter methylation by permeable factors in the transwell system. DBH promoter methylation in the lower layer exposed cells was quantified by MSRE qPCR as described above. ± SEM shown, n = 3, Student's t test, **p = 0.0093. www.nature.com/scientificreports/ In order to examine the epigenetic effects of T. gondii on DBH in neurons, ex vivo experiments were performed with infection of organotypic brain slices of the prefrontal cortex, nucleus accumbens and ventral tegmental areas and the TGS in the neuron population measured, as the percentage of infected neurons during chronic infection is 0.002-0.14% 29,41 . Methylation of the DBH upstream region rose from 29 ± 2.7% in the brain tissue slices to 74 ± 4.6% in the infected slices (p = 0.000051) (Fig. 4A). DNA methylation levels were then analysed in the brains of chronically-infected mice. Neurons were purified from other brain cell types by FACS and DBH methylation was measured. The DBH gene in infected animals was 53 ± 7.7% methylated in neurons compared to 6.3 ± 2.0% in uninfected mice (p = 0.000045, Fig. 4B). For comparison, levels of total genomic DNA methylation were measured (Fig. 4C). No change in global DNA methylation was observed in noradrenergic cells or neurone Slice cultures were either uninfected (light grey, n = 6) or infected with wild-type induced-bradyzoite T. gondii at MOI 5 (dark grey, n = 3) and MOI 7 (black, n = 3). Infection was monitored for 5 days by light microscopy; biological repeats contain 3 slices from the same rat * p = 0.04, ***p = 0.000051 shown. One-way ANOVA, p = 0.0005 Tukey's post hoc test MOI 7 vs uninfected p = 0.0004. (B) Methylation at the DBH promoter region in neurons from uninfected (grey, n = 9) and chronically-infected (black, n = 11) mice. Neurons were enriched from harvested brains by FACS using NeuN antibody and DBH methylation quantified by MSRE-qPCR. p = 0.0004. (C) Total genomic DNA methylation measured by ELISA of both uninfected and infected rat catecholaminergic PC12 cells (n = 3, p = 0.52), human neuronal BE(2)-M17 cell (n = 5, p = 0.59) and neuronal nuclei enriched by FACS from mouse brain tissue for uninfected (n = 6) and chronically-infected mice (n = 3, p = 0.15). No significant change in methylation was identified by Student's t tests, ± SEM shown. (D) Expression of DBH mRNA measured RT-qPCR relative to GAPDH following treatments. Uninfected (grey) and T. gondii-infected (black) noradrenergic PC12 cells treated with trichostatin A (TSA), 5-azacytidine (5-AC) and RG108; n = 5, ** p < 0.01. (E) As in (D) with noradrenergic human BE(2)-M17 cells treated with TSA, 5-AC and RG108; n = 5, *p < 0.05. n = 5. Student's t test, ± SEM shown. (F) 5′ DBH hypermethylation was measured by MSRE-qPCR (as above). Uninfected (grey) and T. gondii-infected (black) PC12 cells were treated with trichostatin A (TSA), 5-azacytidine (5-AC) and RG108; n = 5, **p < 0.01. (G) Uninfected (grey) and infected (black) BE(2)-M17 cells treated as in (F) with TSA, 5-AC and RG108; n = 5, *p < 0.05. Student's t test, ± SEM shown. www.nature.com/scientificreports/ with T. gondii infection, as has previously been found 42 . Hypermethylation of CpG residues upstream of the DBH gene were also found by NGS genomic bisulfite sequencing of infected cultures (Fig. S6). The mechanism responsible for the parasite-induced DNA methylation and chromatin remodelling associated with the silencing was explored by investigating the role of DNA methyltransferase (DNMT) and histone deacetylation in this process. Infected noradrenergic cells were treated with the DNMT inhibitors RG108 and 5-azacytidine (5-AC). Both compounds disrupted the parasite-induced DBH down-regulation and DNA hypermethylation in rat noradrenergic cells, relative to marker (Fig. 4D, F). The DNMT inhibitors similarly blocked the TGS and epigenetic changes in human neuronal cells (Fig. 4E, G). This was not due to parasite sensitivity to the inhibitors since the inhibitors were non-toxic to parasites at concentrations tested (data not shown) and T. gondii lacks 5-methylcytosine 43 . This implies that the DBH silencing involves DNMT. In contrast, the histone deacetylase inhibitor trichostatin A did not abrogate the DBH down-regulation or hypermethylation in infected cultures ( Fig. 4D-G). Hence the mechanism may not involve histone deacetylation or activation was not captured within the experimental timeframe of 5 days and histone deacetylase is active at a different time in the epigenetic modification pathway.

Scientific Reports
To directly compare the epigenetic changes in infected versus uninfected (but infection-exposed) cells in the same culture, populations were enriched for cells containing GFP-expressing T. gondii (GFP+) and GFP− cells. DBH hypermethylation was found in both populations of cells (Fig. 5A, B; ANOVA test, p = 0.0089). Indeed, the GFP(-) cells exhibited DNA methylation levels at least equal to the GFP+ cells. This further supports the supposition that the silencing is spread and direct infection is not required for hypermethylation of the DBH gene in a cell.
In order to observe the induction of DBH hypermethylation in uninfected cells exposed to infected cultures, the methylation status of the DBH upstream region in noradrenergic cells was measured by MSRE-sqPCR. Methylation of the DBH 5' region was 34 ± 4.8% greater (p = 0.0013) in rat cells exposed to infected cells compared to exposure to uninfected cultures (Fig. 5C). Human noradrenergic cells exposed to infected cells also had increased methylation (49 ± 1.9%, p = 0.0003) of the DBH upstream region (Fig. 5D). Hence, both transcriptional down-regulation and promoter hypermethylation are induced in human and rat noradrenergic cells exposed to T. gondii cell cultures.
Based on the above findings, the isolated TINEVs ability to induce epigenetic changes was next investigated. DBH gene promoter methylation, examined using the MSRE-qPCR assay, increased from 16 ± 3.9% to 81 ± 7.9% over the course of the experiment. In a time course of exposure to TINEVs, methylation was not significantly changed after 24 or 48 h of exposure but hypermethylation was observed at 96 h of incubation ( Fig. 5E; ANOVA p = 0.0008). Hence DBH transcriptional down-regulation and chromatin remodelling are induced by EVs from infected cells.
Toxoplasma-induced neuronal EVs contain an antisense DBH lncRNA. We investigated the RNA cargo for association with the TINEV-induced TGS and epigenetic modifications. Preliminarily, the sensitivity of the TINEVs to ultraviolet (UV) irradiation was tested to assess whether an RNA component could be involved 44 . TINEVs treated with UV radiation no longer induced hypermethylation of the DBH promoter (Fig.  S7). Although, it is possible that the UV radiation inactivated proteins.
With the specificity of the differential gene expression and the role of non-coding RNAs (ncRNA) in regulating gene expression, we explored the potential presence of a long non-coding RNA (lncRNA). LncRNAs have roles in neuronal gene expression such as several highly specific antisense transcripts [45][46][47][48] . EVs have been found to contain miRNAs and lncRNAs, although their functional significance is still unclear. With the specificity of the DBH down-regulation in T. gondii infection and its magnitude with TINEV treatment, the identification of a long non-coding RNA (lncRNA) was investigated 45,49 . A panel of primers walking upstream stepwise from the DBH coding region were used to screen for an antisense RNA 37 . These identified a lncRNA in infected cultures (Fig. S8). RNA purified from TINEV preparations were found to contain the DBH antisense lncRNA ( Fig. 6A-C, Fig. S9, S10). Although the lncRNA location internally as EV cargo is unconfirmed, it is unclear of the functional significance of the location 50 . The lncRNA is complementary to the DBH upstream region containing cis-regulatory elements and crosses the transcription start site. As a positive control for samples, miR-21 miRNA served to confirm RNA quality purified from the TINEVs 51 . Intriguingly, the timeframe observed for the DBH hypermethylation in this study (Fig. 5E) is similar to dynamic DNA methylation changes observed in cells treated with promoter targeted antisense RNA 52 . The observations above represent the first study to show a specific and functionally-relevant lncRNA to be transmitted from one neuronal cell to another regulating neurotransmission 53 .
The DBH antisense lncRNA was found to contain the conserved U1 snRNP recognition site that is a consensus sequence identified in chromatin-bound lncRNAs 54 . Further, predicted secondary structure analysis places the U1 snRNP recognition site in a loop region in minimum free energy secondary structures of DBH antisense lncRNA (Fig. 6D). In the published study, the recognition site needs to be in a loop region to allow binding of U1 snRNP to the lncRNA to promote chromatin interaction. Future experiments will further investigate this novel induced lncRNA.

Discussion
Intracellular infection can manipulate gene expression not only within the host cell but signalling to other cells. In this study it was found that T. gondii infection resulted in the release of EVs from noradrenergic cells that specifically induced gene silencing and DNA hypermethylation in vitro and in the brain. Many cells of the CNS release EVs including astrocytes, glia, oligodendrocytes, Schwann cells and neurons [55][56][57] . EV secretion is emerging as an important mechanism of cellular communication within the CNS 58  www.nature.com/scientificreports/ cultures and delivered into the brain induced DBH down-regulation in neurons. EVs are continuously released in the brain limiting our ability to detect and isolate those induced by the 0.002-0.14% of cells that are infected during chronic T. gondii infection and restricted by the current lack of biomarkers for TINEVs. The findings presented here provide a mechanism that can resolve the enigma of an observable decrease in NE when only a small percentage of brain cells are infected. We propose a model of T. gondii inducing TINEV release with host lncRNAs that modulate the host environment 59 . Studies of extracellular vesicles secreted from pathogen-infected cells, including the apicomplexan parasites, have reported altering the host immune response [60][61][62][63][64] . For example, cells infected with Epstein-Barr virus release EVs containing viral miRNAs that can downregulate cytokine expression in uninfected monocyte-derived dendritic cells. Human miRNAs complexed with Argonaute 2 are released in EVs from Plasmodium-infected red blood cells and induce pro-inflammatory cytokines in macrophages and endothelial cells. The findings reported here, in contrast, demonstrate a role for TINEVs in altering neurophysiology. TINEVs spreading to uninfected neurons carried host mammalian lncRNA, regulated gene expression, and modified chromatin. Intriguingly, NE modulates CNS cytokine release and hence the resultant NE decrease from DBH down-regulation will also www.nature.com/scientificreports/ affect the neuroimmune response. Host hypomethylation of the mammalian arginine vasopressin promoter in the amygdala of T. gondii-infected rodents has also been observed 42 . EV-miRNAs have been reported that alter DNA methylation in surrounding cells 63 . Here, we identified a natural mammalian antisense lncRNA in TINEV preparations. The lncRNA is complimentary to the DBH 5'UTR and contains a consensus sequence for chromatin localization. It remains possible that DBH down-regulation is imposed by cell-autonomous immunity in noradrenergic cells by another component of TINEVs that has yet to be defined. Chromatin localisation of the lncRNA may occur via U1 snRNP binding at the transcriptionally active DBH gene and interacting with RNA polymerase II to disrupt transcription and facilitate DNA methylation. In this study, diffusible products of infected fibroblasts including EVs did not induce DBH down-regulation (Fig. 4F). In the future it will be interesting to assess the intercellular crosstalk from infected neurons to different brain cell types and to examine how T. gondii induces host cells to express and package selective lncRNAs.
Evidence is rapidly accumulating to support the role of EVs in the regulation of neurogenesis, neuronal connectivity and neuroimmune communication 44,65 . The findings in this study describe a novel form of neurotransmission regulation that may contribute to maintaining chronic infection. We identified a natural antisense lncRNA in TINEV preparations that specifically altered neurotransmitter signalling. In a recent study, reduced www.nature.com/scientificreports/ CNS noradrenergic signalling during chronic T. gondii infection correlated with noradrenergic-linked behaviour changes 11,66 . Chronic infection with bradyzoite-infected neurons permits continuous delivery of TINEVs into the brain. In this study, TINEVs delivered by intracranial injection twice daily for 3-days down-regulated DBH.
As behaviour changes are measured in rodents during the 4th to 6th week of infection, a sustained supply of TINEVs (or the antisense lncRNA) is necessary for comparative behaviour tests. Also, reduced DBH has been found to elevate dopamine release from LC neurons (such as those modulating learning and memory) 7,67 which could explain increased dopamine signalling that has been observed with T. gondii infection 68 . The neuroimmune system may also be affected by EVs. Changes in NE levels can alter cytokine responses via adrenergic receptors on astrocytes and microglia. Indeed, treatment with an adrenergic receptor agonist reversed the increased locomotion of infected mice 3,69 . Finding that EVs can induce TGS and modify DNA methylation neurons may represent a mechanism, beyond parasitic machinery, in innate neurophysiological function and enhance our understanding of the role of transcriptional regulation and dynamic DNA methylation in memory and learning.

Infection of PC12 and BE(2)-M17 cells.
Cells were cultured in multi-well plates at a density of 5 × 10 4 cells/ml. When stated, transwell plates (Nunc™ cell culture inserts, Thermo Scientific) were used, with cells plated at 2.5 × 10 4 cells/ml. Following 24 h of growth on 6-well plates, cells were infected with induced Prugniaud parasites in upper-wells, maintaining a multiplicity of infection (MOI) of 1. Cells were harvested immediately following infection (day 0) and after 3 and 5 days of infection for downstream processing. Mock-infection was performed using heat-killed T. gondii parasites subjected to 80 °C for 10 min. The cultures were monitored daily by light microscopy.

Treatment of cells with purified TINEVs.
EVs were extracted and purified from cell medium of uninfected and infected cells in exosome-depleted media, as described below. PC12 cells plated on 6-well plates and grown to a density of 2 × 10 4 cells/ml were treated with EVs at a 10:1 culture media concentration equivalent (ie. 1 ml of PC12 cell culture treated with EVs purified from 10 ml of culture). Cells were treated every 24 h. DNA and RNA were harvested 24 h after the last treatment.

Rodent experiments.
Nine-week-old wild-type Sprague Dawley male rats weighing 245 ± 15 g were obtained from Charles River Laboratories (Margate, UK) and were used in line with the United Kingdom Animals (Scientific Procedures) Act 1986 and ethical standards set by the University of Leeds Ethical Review Committee and in accordance with the ARRIVE guidelines. Animals were housed individually and maintained on a 12-h light-dark cycle with access to food and water ad libitum. Rats were monitored for health and fitness parameters of weight and food intake as shown in the supplemental data and the one that did not recover after surgery (lost more than 10% of their body weight) was not used in the experiments. Animals were randomly www.nature.com/scientificreports/ assigned from a total of 11 rats; 10 were used in this study. Animal care was blinded with investigators aware of the treatments. Animals were treated twice daily for 3 days. The outcome was measured by expression of DBH and compared by Student's t-test. Six male Lister Hood rats (Harlan Ltd, UK), 9-weeks old and weighing circa 300 g were infected and monitored as previously described 11 . Rats were housed in individual cages and maintained on a standard light-dark cycle with access to chow diet and water ad libitum. Animals were infected by intraperitoneal (IP) injection with tachyzoites. Throughout the experiments animals were monitored for illness or weight loss (more than 10%) and sacrificed 5-6 months post-infection. Brains were snap-frozen for cryosectioning and RNA extraction.
(BALB/cAnNCrl × C57BL/6NCrl) F1 generation male mice (29 mice, no exclusions) were housed up to five to a cage and maintained on a standard light-dark cycle with access to chow diet and water ad libitum. Throughout the experiments animals were monitored for illness or weight loss (more than 10%) and sacrificed 4-6 weeks after infection in the University of Leeds research animal facility with procedures approved by the University Animal Care and Use Committee and following Home Office, HSE, regulations and guidelines for Animals (Scientific Procedures) Act 1986 published in 2014 and in accordance with ARRIVE guidelines with considerations of the replacement, reduction, and refinement in the use of animals for research. The mouse infections, brain dissection and frozen cryostat sectioning was performed as described 11 . Organotypic brain slice cultures. Sprague-Dawley rat pups (one litter) from females housed individually with ad libitum chow diet and water at postnatal days 3-4 were anesthetized as approved, following the Kyoto University Animal Research Committee approved protocols in the Kaneko Laboratory at Kyoto University. Sectioning was performed as described 41 . Brain was removed and separated into two hemispheres. Coronal sections (350 μm thickness) were prepared under sterile conditions. Slices were dissected to include the ventral tegmental area (VTA). These slice cultures were placed on 30 mm insert membranes (Millicell 0.4 μm; Millipore). Slice culture medium contained RPMI supplemented with 10% horse serum (GIBCO, USA), 5% FBS and additional 6.5 mg/ml glucose and 2 mM l-glutamine. The brain slice cultures were cultured at 37 °C in a 5% CO 2 for 15 days after dissection prior to use in experiments.

Extracellular vesicle intracerebral treatment.
Intracerebral injection into adult rats followed previously described experimental procedures 73 . TINEVs extracted and purified from rat catecholaminergic PC12 cells as described below were used to treat rats. Rats were stereotactically (David Kopf Instruments) implanted with indwelling bilateral cannula targeting the locus coeruleus. After one week of recovery, implanted rats received a 2 μl infusion twice a day for 3 days with TINEVs (4 μg protein) isolated from infected PC12 cells. EVs from uninfected PC12 cells served as a control. On the fourth day rats were anaesthetized and received an injection 3 μl bromophenol blue through each side of the bilateral DVC cannula (to verify their placement) prior to harvesting brain tissue. Brain tissue was dissected into prefrontal cortex, midbrain, and hindbrain regions for processing for RNA.
Fluorescence-assisted cell sorting (FACS) of mouse neurons. Mouse brain samples were processed as described 74 . Briefly, brains were suspended in lysis buffer (0.32 M sucrose; 0.1 mM ethylenediaminetetraacetic acid; 5 mM CaCl 2 ; 3 mM Mg(Acetate) 2 ; 10 mM Tris-HCl pH 8; 1 mM dithiothreitol; and 0.1% Triton X-100) and processed using a 15 ml glass dounce homogenizer (Wheaton, UK) in 5-10 strokes on ice. Samples were centrifuged at 100,000xg with a two-step sucrose gradient to purify nuclei. Nuclei were stained with primary antibody NeuN (Abcam, EPR12763 1:250) and secondary antibody Alexa-488 (Abcam, ab150077, 1:1000). All nuclei were also stained with Hoechst 33342 (SIGMA, USA). Samples were sorted using the Becton Dickinson FACSAria II system. Extracellular vesicle characterization. Extracellular vesicle purification. TINEVs were isolated from media by ultracentrifugation as described 75,76 . For all experiments, EVs were isolated in parallel from uninfected cells. Briefly, one day prior to harvest, complete media was replaced with media containing exosome-depleted FBS (Systems Bioscience, CA). Cell cultures of uninfected and infected cells (five days following infection) were harvested and centrifuged at 3000×g for 10 min at 4 °C. The supernatant was isolated and centrifuged at 160,000×g for 2 h in a Type 60 Ti fixed angle rotor at 4 °C. The pellet was resuspended in one ml of 90% sucrose. Eleven layers of one ml of sucrose 70%-10% w/v were then added and centrifuged at 70,000×g for 16 h at 4 °C in a Type 60 Ti fixed angle rotor. Supernatant was collected in two ml fractions. PBS (8 ml) was added to the fraction 1.1-1.2 g/ml sucrose with a density corresponding to that of EVs and centrifuged at 160,000xg for 70 min at 4 °C to recover purified EVs. Methods and data on purification and characterization were input to the EV-TRACK knowledgebase (EV-TRACK ID: EV220363) (https:// evtra ck. org/). UV treatment. EVs were isolated as described. Prior to UV treatment, DBH down-regulation by TINEVs was verified following incubation of noradrenergic cells for 24 h. TINEVs were exposed or mock exposed to UV light at 302 nm for 5 min at 4 °C using a transilluminator (Syngene, Cambridge). Dot blotting. Dot blotting was performed using the Exo-Check Exosome Antibody Array (Systems Bioscience, CA) as per manufacturer's instructions. Briefly, 500 μg protein equivalent of purified EVs isolated from PC12 culture were lysed and ligated to horseradish peroxidase (HRP) overnight at 4 °C. Ligated protein was then incubated with the antibody membrane for two hours at room temperature. After three washes SuperSignal West Femto Chemiluminescent Substrate kit (Thermo Scientific, UK) was used as per manufacturer's instruc- Transmission electron microscopy (TEM). EVs were purified as described above. Electron microscopy was performed as described 77 . Freshly isolated EVs were resuspended in cold Dulbecco Phosphate-Buffered Saline (DPBS) containing 2% paraformaldehyde. EVs were mounted on copper grids, fixed with 1% glutaraldehyde in cold DPBS for 5 min at room temperature, negative stain was performed with uranyl-oxalate solution at pH 7 for 5 min, and embedded with methyl cellulose-UA for 10 min at 4 °C. Excess cellulose was removed, and samples were dried for permanent preservation. Electron microscopy was performed in the University of Leeds facility using a Titan Krios 2 electron microscope. Analysis and extracellular vesicle diameter were measured using ImageJ software.

Molecular techniques. DNA extraction.
Classic phenol-chloroform method extraction was performed as described in 78  Nuclear run-on. Nuclear run-on was performed as described 79  Methylation sensitive restriction enzyme (MSRE) quantitative PCR. DNA was extracted from cell cultures and isolated as previously described. DNA concentrations were confirmed using a Nanodrop spectrophotometer. Samples were divided into reference and test samples. Test samples were digested with the highfidelity methylation sensitive restriction enzymes (MSRE) HpaII, MaeII and SmaI (TaiI) in the Tango buffer (Thermo Fisher Scientific, USA). These MSREs were chosen based on the restriction enzymes sites present in the DBH upstream region. Reference samples were mock digested. Digestion was performed by heating to 25 °C for 30 min, 37° for 30 min and 65° for 30 min. RT-qPCR was then performed. Percentage methylation calculated based on the difference in Cq values between test and reference samples. Digestion was confirmed with methylated and unmethylated human DNA standards provided with the OneStep qMethyl kit (Zymo) as per manufacturer's instructions. Additionally, primers and MSRE digestion was confirmed with MSRE-PCR. qPCR was performed using SYBR®Green Real-Time PCR Master Mix (Thermo Fisher).
Directional RT-PCR for DBH lncRNA screening. Three single-stranded forward (i.e. sense) directional primers were designed to detect antisense RNA and are labelled RT-Primer 1, RT-Primer 2, and RT-Primer 3 (Extended Data Fig. 6A). The RT-designed primers are located (1) in the first exon, (2) near the transcription start site, and (3) − 378 bp upstream of the DBH coding sequence. Four pairs of PCR primers were then designed to amplify products from the RT primer synthesized-template to screen for the presence of antisense lncRNA (Extended Data Fig. 6A). A list of primers used is shown, Supplementary Table 2.
Total RNA was extracted from T. gondii infected-PC12 cells that were harvested on day five post-infection using the Direct-zol RNA MiniPrep Plus kit (Zymo Research, USA) according to the manufacturer's instructions. DNase I treatment was implemented as suggested by the manufacturer. In addition, to ensure that all trace amounts of DNA contamination were removed, the eluted RNA samples were treated again with DNase enzyme using TURBO DNA-free™ kit (Invitrogen, USA) according to the manufacturer's instructions as in earlier studies 37  www.nature.com/scientificreports/ with RT-primer 1 to 3 with a Maxima H Minus First Strand cDNA synthesis kit (Thermo Scientific, USA) following the manufacturer's instructions. For each of the samples, there were several controls including priming the cDNA synthesis with random hexamer primer, a negative control reaction to assess the gDNA contamination in the RNA sample containing all components for RT-PCR except the Maxima H Minus enzyme mix, and no template control to assess reagent contamination. The mixtures were incubated as follows: 25 °C for 10 min, 50 °C for 30 min, 65 °C for 20 min and then the reaction was terminated by heating at 85 °C for 5 min. The reaction products served as templates for PCR with GoTaq ® G2 Hot Start Master Mix (Promega, USA), 300 nM forward primer, 300 nM reverse primer, and 2 μl template DNA. Thermal cycling was 3 min at 95 °C, followed by 30 cycles of 95 °C for 30 s, 57 °C for 30 s, 72 °C for 20 s and final termination at 72 °C for 5 min in a thermocycler (Applied Biosystems, USA). All PCR products were resolved and visualized by 1.5% to 2% w/v agarose gel electrophoresis. For DNA sequencing, the specific band was gel-excised and gel-extracted using QIAQuick Gel Extraction Kit (Qiagen, Germany). The eluted DNA concentrations were then measured by the NanoDrop spectrophotometer (Thermo Fisher Scientific, UK). The PCR products were subcloned into a TOPO TA cloning vector (PCRTM4-TOPO-Invitrogen TA cloning kit, Thermo Fisher Scientific, UK) and transformed into XL-10 Gold ultracompetent cells (Agilent Technologies, USA). Two clones bearing inserts of each sample were sent for Sanger Sequencing to Genewiz, UK.
Global methylation. Global methylation was measured using a colourimetric, ELISA adapted method,

Availability of data and materials
All data generated and analysed during this study are included in this published article [and its supplementary information files]. The raw datasets from the current study are available from the corresponding author on request.